The flavivirus genus incorporates over 60 closely related viruses including several human pathogens of the global and local epidemiological importance. Virions are composed of three structural proteins designated capsid (“C”), membrane (“M”), and envelope (“E”). Immature flavivirions found in infected cells contain pre-membrane (“prM”) protein, which is a precursor to the M protein. Immature virions contain prM-E heterodimers composing the virion envelope. The prM protein serves as a chaperone for slowly folding E, prevents E from pH-mediated irreversible rearrangement during transport, and is cleaved prior to virion release. Flavivirus-infected cells release non-infectious subviral particles containing only envelope proteins prM and E. These can be generated by expression of flavivirus prM-E cassettes. Their assembly pathway—intracellular transport, carbohydrate processing, maturation, prM cleavage, and secretion—resembles that of infectious virions.
The E protein comprises a long ectodomain followed by a stem-anchor region. Three-dimensional structures of the flavivirus E protein ectodomain (about 400 amino acids, excluding the carboxy terminal stem and transmembrane domains) and its dimeric and trimeric forms have been solved for E proteins of tick-borne encephalitis and dengue viruses, both in the prefusion and postfusion conformations. See Bressanelli et al., Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation, EMBO J. 12 1-12 (2004); Modis et al., A ligand-binding pocket in the dengue virus envelope glycoprotein, Proc Natl Acad Sci USA 100 6986-6991 (2003) Epub May 20, 2003; Modis et al., Structure of the dengue virus envelope protein after membrane fusion, Nature 427 313-319 (2004); Modis et al., Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein, J Virol 79 1223-1231 (2005); and Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution, Nature 375 291-298 (1995), which are incorporated by reference. The ectodomain forms an elongated dimer that is oriented parallel to the viral membrane (see FIG. 1, the top view of a NY99 E400 model derived by homology modeling). In the head-to-tail dimer, each monomer is composed of domains I, II, and III. Monomer contacts in the dimer are not contiguous along the whole length of the molecule. There are two holes along the dimer axis that occupy the place of cleaved prM (see Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution, Nature 375 291-298 (1995)). Beyond two short α-helices in domain II, β-strands are predominant throughout the molecule.
Each of the centrally located N-terminal domains I contains two disulfide bridges and carries a single carbohydrate side chain that shields the fusion peptide located on the tip of domain II and contributes to overall stability of the dimer (see Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution, Nature 375 291-298 (1995)). Domain II, or the dimerization domain, has an elongated finger-like structure and is involved in monomer-to-monomer interaction at two distinct loci. The distal loop is stabilized by three disulfide bridges and forms the tip that holds the fusion peptide, which fits into a hydrophobic pocket provided by domains I and III of the second monomer. This contact is largely nonpolar and is composed of residues from domains I and III on one subunit and the tip of domain II on the other. The contact at the center, where two prominent α-helices can be seen, mostly involves hydrophilic side chains of domain II only. Domain III contains the C terminus and in the virion is connected to the stem followed by the transmembrane region that anchors the monomer in the membrane.
Despite the divergence in amino acid sequences of the E proteins of different flaviviruses, the 12 cysteine residues are absolutely conserved between species. These form six disulfide bridges in the West Nile virus E protein (see Nowak et al., Analysis of disulfides present in the membrane proteins of the West Nile flavivirus, Virology 156 127-137 (1987)) and were found at the expected positions in the X-ray structures of all E proteins determined to date. This strongly supports the current understanding that the overall structural organization and folding are similar for E proteins of all flaviviruses.
Exposure to acidic pH leads to dramatic rearrangement of the virion organization accompanied by inactivation of biological activities such as infectivity, membrane binding, and fusion. Induced changes are a crucial component of the fusion process during virus entry. See Corver et al., Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system, Virology 269 37-46 (2000); Heinz et al., The machinery for flavivirus fusion with host cell membranes, Curr Opin Microbiol 4 450-455 (2001); and Stiasny et al., Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH, J Virol 76 3784-3790 (2002). The mechanism of pH-induced fusion mediated by the E protein involves rearrangement of E from the dimeric to a trimeric form (see Stiasny et al., Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus, J Virol 70 8142-8147 (1996)). Formation of fusogenic E trimers is a two-step process, in which dimers first dissociate under influence of low pH and then re-associate forming trimers. The two-step model has been first obtained in studies of the E-400 ectodomain. In absence of the stem-anchor region exposure to acidic pH causes reversible dissociation of the dimer that does not lead to trimerization. Further studies demonstrated the functional role of the stem-anchor region (about amino acids 400-449) for the low-pH-induced irreversible conversion of the dimer to the trimer in solution (see Allison et al., Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E, J Virol 73 5605-5612 (1999)). The irreversible change from dimers to trimers induced by low pH suggests that in virions E exists as a metastable dimer and changes to a more stable trimer when the appropriate trigger (in this case low pH) is applied. It was shown that the trimeric form of E is more stable to thermal denaturation than the dimeric form. In contrast to class I fusion proteins, however, such transition to the more stable conformation state cannot be induced by thermal treatment, which only leads to the denaturation of E (see Stiasny et al., Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus, J Virol 75 7392-7398 (2001)). This suggests that protonation of the native E dimer is indispensable for generating a monomeric intermediate structure that is required for the formation of the energetically more stable final trimeric form (see Heinz et al., Flavivirus structure and membrane fusion, Adv Virus Res 59 63-97 (2003)).
For a number of flaviviruses neurovirulent and neuroinvasive phenotypes have been associated with envelope proteins. See Cecilia et al., Nucleotide changes responsible for loss of neuro invasiveness in Japanese encephalitis virus neutralization-resistant mutants, Virology 181 70-71 (1991); Chambers et al., Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties, J Virol 73 3095-3101 (1999); Gualano et al., Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA, J Gen Virol 79 437-446 (1998); Hasegawa et al., Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice, Virology 191 158-165 (1992); Holzmann et al., A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model, J Virol 64 5156-5159 (1990); Holzmann et al., Characterization of monoclonal antibody-escape mutants of tick-borne encephalitis virus with reduced neuro invasiveness in mice, J Gen Virol 78 31-37 (1997); Jiang et al., Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence, J Gen Virol 74 931-935 (1993); McMinn, The molecular basis of virulence of the encephalitogenic Flaviviruses, J Gen Virol 78 2711-2722 (1997); Pletnev et al., Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses, Proc Natl Acad Sci USA 89 10532-10536 (1992); and Pletnev et al., Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice, J Virol 67 4956-4963 (1993), which are all incorporated by reference. However, mutations in other parts of the genome were also implicated for loss/acquisition of neurovirulence. See Butrapet et al., Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5′ noncoding region and nonstructural proteins 1 and 3, J Virol 74 3011-3019 (2000); Duarte dos Santos et al., Determinants in the Envelope E Protein and Viral RNA Helicase NS3 That Influence the Induction of Apoptosis in Response to Infection with Dengue Type 1 Virus, Virology 274 292-308 (2000); Dunster et al., Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus, Virology 261 309-318 (1999); Muylaert et al., Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence, Virology 222 159-168 (1996); Ni et al., Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14, J Gen Virol 76 409-413 (1995); and Xie et al., Yellow fever 17D vaccine virus isolated from healthy vaccinees accumulates very few mutations, Virus Res 55 93-99 (1998), which are all incorporated by reference. Attenuation resulting from mutations in protein E is most extensively studied with a live attenuated JE vaccine SA14-14-2, for which 9 amino acid differences have been identified in the E protein that distinguish the attenuated vaccine virus from its virulent parent SA14. The dominant attenuating effect is associated with a E138K mutation located at the so-called “hinge” region interfacing domains I and II (see Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution, Nature 375 291-298 (1995)). The hinge locus is believed to play a crucial role in dimer-to-trimer transition of the E protein associated with virus entry. Modifications within this region modulate virulence of flaviviruses in mice (see Cecilia et al., Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants, Virology 181 70-71 (1991); Gualano et al., Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA, J Gen Virol 79 437-446 (1998); Hasegawa et al., Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice, Virology 191 158-165 (1992); Hurrelbrink et al., Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein, J Virol 75 7692-7702 (2001); McMinn et al., Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice, Virology 211 10-20 (1995); and Sumiyoshi et al., Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA, J Infect Dis 171 1144-1151 (1995), which are all incorporated by reference). Additional loci important for attenuation or reversion to virulence were defined at positions 176/177 and 264/279 in E and are also present in SA14-14-2. The former is located in the central domain undergoing changes during acid-mediated reorganization of E to fusion competent trimers during virus entry (see Bressanelli et al., Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation, EMBO J. 12 1-12 (2004)). The latter locus is also located in the hinge region and may functionally cooperate with the locus defined by the E138K mutation, since mutations involving nearby positions impair hemagglutination and fusion properties of E and reduce neuroinvasiveness in mice (see Hurrelbrink et al., Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein, J Virol 75 7692-7702 (2001) and McMinn et al., Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice, Virology 211 10-20 (1995)). The last cluster of mutations present in SA14-14-2 is located to the domain III and stem-anchor region of the E protein, which are important for virus attachment to cells and for interaction with prM. Mutations around position 315 resulted in altered virus tropism and changes in virulence (see Jennings et al., Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis, J Infect Dis 169 512-518 (1994); Jiang et al., Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence, J Gen Virol 74 931-935 (1993); Ni et al., Attenuation of Japanese encephalitis virus by selection of its mouse brain membrane receptor preparation escape variants, Virology 241 30-36 (1998); and Ryman et al., Mutation in a 17D-204 vaccine substrain-specific envelope protein epitope alters the pathogenesis of yellow fever virus in mice, Virology 244 59-65 (1998)). The integrity of the stem-anchor region is also required for stability of the prM-E heterodimer (see Allison et al., Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E, J Virol 73 5605-5612 (1999)). The only amino acid change in SA14-14-2 that is found in the distal monomer contact interface (see FIG. 1) involves a L107F substitution in the highly conserved fusion loop (amino acids 98-110). In the vast majority of flaviviruses, this position is occupied by Leu, with only two known exceptions of the Phe occurrence in Powassan and deer tick flaviviruses that are substantially less virulent American relatives of TBE virus. Reversion of this mutation to Leu was associated with only partial reversion to the neurovirulent phenotype (see Arroyo et al., Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE), J Virol 75 934-942 (2001)) indicating that only minor attenuation changes are tolerated at this locus. Lack of other known mutations at either distal or central contact interfaces indicates the existence of a strong selective pressure against changes influencing dimer formation. This agrees with the importance of this interface both for virion assembly/maturation at the end of the viral infectious cycle and for functional disassembly during the initial phase of the next reproductive cycle.